Hedgehog (HH) signaling is essential for the specification and growth of a wide variety of organs and tissues during embryonic development and has a continued role in the maintenance and regeneration of adult tissues. Deregulation of the HH signaling pathway can lead to numerous developmental disorders, birth defects, and childhood and adult cancers. Neuropilins (NRPs), canonical receptors for the Semaphorin family of axon guidance molecules, can positively regulate HH pathway function through an unknown mechanism (Hillman et. al., 2011). NRPs require Plexin (PLXN) coreceptors to transduce semaphorin signals, but the role of PLXNs in HH signaling has yet to be explored. Using cell signaling assays, I have confirmed that NRPs promote HH signaling in NIH/3T3 fibroblasts. Moreover, my preliminary data indicate that NRPs traffic through the primary cilium, a key platform for HH signal transduction. My data also suggest that both NRP-mediated promotion of HH signal transduction and NRP ciliary localization require the NRP cytoplasmic domain. I aim to elucidate the role of the NRP cytoplasmic domain in HH signal transduction as well as to determine the requirement for the NRP extracellular domains through a systematic structure-function approach. Strikingly, I have also observed that several PLXNs promote HH signaling. My initial findings suggest that only a subset of PLXNs promote HH signaling, specifically the PLXNA family subgroup, and that PLXNs require an intact transmembrane and cytoplasmic domain for this promotion. I propose to identify the specific PLXN subgroups involved in HH signaling and further characterize their mechanism of action in vivo. Importantly, I have found that PlxnA1;PlxnA2 double mutant mice exhibit sternal and cervical spine midline defects reminiscent of HH- dependent phenotypes, strongly implicating PLXNs as novel HH regulators. Overall, my data provide compelling evidence for crosstalk between multiple members of the Semaphorin and HH signaling pathways, which are coexpressed spatially and temporally during development. I hypothesize that the semaphorin receptors, NRPs and PLXNs, are novel HH pathway components that are required for proper HH signal transduction during embryonic development. This proposal will uncover fundamental insights into HH regulation at the cell surface and have therapeutic implications for a variety of HH-dependent disorders.